Abstract
Objectives
An easy-to-operate method of using R-ω-transaminase has been developed by fusing it to an elastin-like polypeptide and forming a complex with D-amino acid oxidase.
Results
R-ω-Transaminase (R-ω-TA) was fused to an elastin-like polypeptide (ELP) through genetic engineering of the enzyme. The enzyme was purified through reversible phase transition. For the single-enzyme system, in the reaction media, ELP-R-ω-TA self-assembled and formed enzyme clusters of micrometer size, and the substrate, (R)-1-phenylethylamine, also formed droplets of micrometer size. Intimate contact of the enzyme clusters and the substrate droplets provided a microenvironment of high substrate concentration close to the enzyme, facilitating the diffusion of substrate molecules into the active sites. For the two-enzyme system, ELP-R-ω-TA and ELP-fusion D-amino acid oxidase assembled to form two-enzyme complexes, forming clusters with a size much larger size than that of single enzymes. The efficiency of the combined enzymes for producing the product was 99.6 %.
Conclusions
The two-enzyme complexes significantly improved the catalytic efficiency. Potentially, the two enzymes forming complex clusters can facilitate the immobilization of the two enzymes together through non covalent methods by entrapping in porous supports.
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Acknowledgments
This work was supported by the National Science Foundation of China (21376021, 21576018) and the National Hi-tech R&D Program (2014AA022003).
Supporting Information
Additional experimental details: Gene construction and cloning of an elastin-like polypeptide (ELP); Expression of ELP fusion D-amino acid oxidase.
Supplementary Table 1—R-ω-transaminase gene sequence and primers.
Supplementary Table 2—ELP monomer.
Supplementary Table 3—DAAO gene sequence and primers.
Supplementary Fig. 1—Construction of the plasmid. pET28a/(VPGXG)100/R-ω-TA in Escherichia coli.
Supplementary Fig. 2—Schematic presentation for the purification of ELP-R-ω-TA.
Supplementary Fig. 3—Conversion of (R)-1-phenylethylamine(30 mM) catalyzed by ELP-R-ω-TA and R-ω-TA.
Supplementary Fig. 4—(a): Analysis of ELP-DAAO by SDS-PAGE; (b) confocal image of ELP-DAAO.
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Gao, Q., Fu, Y., Peng, Y. et al. Genetically-modified R-ω-transaminase: purification and self-assembly facilitating interaction with substrate droplets. Biotechnol Lett 38, 489–494 (2016). https://doi.org/10.1007/s10529-015-1998-7
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DOI: https://doi.org/10.1007/s10529-015-1998-7